We will spend the next grant period on epigenetic marks in lung cancer that will build on our previous work on unmasking promoter methylation (Aim 3). We will extend our promoter work to extensively study hypomethylated promoters with a subsequent increase in gene expression for incorporation into EDRN collaborations that focus on RNA or protein (Aim 2). We will assess global methylation status in lung cancer cell lines and primary lung tumors and use new tiled arrays to identify genome-wide regions of methylation (Aim 1). These studies will thus be complementary to our ongoing studies using pharmacologic unmasking and will allow direct comparison between genes methylated in tumors of unclear significance and those that are reactivated after 5-AZA treatment, suggesting a more functional role in tumor progression. Once identified, we will use the assays we developed including MSP and QMSP to test regions of promoter hypermethylation to develop measurable biomarkers. These assays will allow continuing to develop highly specific and sensitive tests to detect tumor cells in various bodily fluids such as serum, plasma, sputum, and BAL fluid for early detection and diagnosis. We have also designed a novel QD-FRET nanoprobe technology for detecting nucleic acids and an additional approach here will be to build these probes for specific methylation markers we discover and validate. The proposed integrated program has the ability to achieve this goal because it optimally applies existing investigator talent, achievement, and interest using the most promising current research directions in epigenetics.